Apoptotically active peptides

ABSTRACT

Specific amino acid sequences and peptides and/or peptide mimetics deducted therefrom influencing apoptosis, and the use thereof for the production of pharmaceuticals as diagnostic tools are shown.

The present invention relates to apoptotically active peptides and theuse thereof for the production of pharmaceuticals.

Apoptosis is a genetically encoded suicide program which underparticular physiologic or pathologic conditions is induced in eucaryotecells. The induction of apoptosis has to be controlled extremelyprecisely since hyperactivity can lead to degenerative disorders.Reduced apoptosis induction, on the other hand, can contribute to tumorprogression.

Various low-molecular inductors of apoptosis have already beendescribed. A significant class are tumor cytostatica. In most of thecases, it is, however, not clear in which way these cytostatica or othersubstances can induce apoptosis.

Induction of apoptosis can for instance result via a series of so-calleddeath receptors, i.e. receptors including a “Death Domain” (DD) such asCD95, TNF-RI, DR3, DR4 or DR5, which after binding of their ligandsinduce apoptosis signal ways. The CD95 receptor, for instance, interactsafter binding of the CD95 ligand with the adapter protein FADD/MORT1whereby “recruitment” and activation of the protease FLICE/CASpase-8 areinduced at the DISC, the “Death Inducing Signaling Complex”. FADD andFLICE each include “Death Effector Domains” (DED). Induction ofapopotsis from outside via these signal ways is possible for instance bythe addition of cell poisons (cytotoxic substances), by irradiation,viruses, withdrawal of growth factors or mechanical cell injure. Thesepossibilities of apoptosis induction, however, are accompanied byspecial disadvantages. Addition of cell poisons, such as cytostatica, orirradiation in case of cancer cells leads to resistance development and,moreover, to damage of normal cells in which, originally, no apoptosiswas to be triggered.

In general, induction of apoptosis is suggested for instance for thetreatment of cancer, for avoiding angio-genetic processes etc. Whileinductors have here been described they still have a series ofdisadvantages. Cytostatica for instance generate heavy side-effects.

However, pathologic states are being discussed as well, whereinapoptosis has negative effects and for the treatment of which apoptosisshould be inhibited.

An example of such a disease is arteriosclerosis. In particular, theinventors were earlier able to show that particularly in the area of thearteriosclerotic plaques, apoptotic cells (particularly endothelialcells, smooth muscle cells) come up and that such coming up is amplifiedby disturbed flow conditions, i.e. is flow-dependent (Freyberg et al.,BBRC, 286, 141 149, 2001). Furthermore, the inventors could show thatsubstances inhibiting the binding of TSP-1 to IAP and/or α_(v)β₃ canalso inhibit flow-dependent apoptosis.

Further diseases discussed in connection with increased up-comingapoptosis are AIDS and Alzheimer's disease.

Also in wound healing, apoptosis of participating cells such asfibroblasts, muscle cells and endothelial cells, plays a significantrole. Inhibition of apoptosis should, therefore have a favorableinfluence on wound healing.

There exists, therefore, a high need for substances having a positive ora negative influence on apoptosis. In particular, it would be favorableto be able to inhibit apoptosis which is flow-dependent and causallyrelated to arteriosclerosis. There is, furthermore, a high need forpharmaceutical formulations comprising substances that can be used fortreating states wherein inhibition or induction of apoptosis isindicated, particularly for the treatment of arteriosclerosis, but alsoof AIDS. Alzheimer's disease and cancer. In this connection, suchsubstances should, if possible, be of lowest molecular weight and/or besmall peptides in order to ensure good biological availability.

It was, therefore, the object of the present invention to provideapoptotically active substances, preferably peptides. In particular, itwas a further object of the present invention to provide substances,preferably peptides, which can inhibit flow-dependent apoptosis of theendothelial cells induced by TSP-1. It was a further object of thepresent invention to provide pharmaceutical preparations by whichdiseases such as AIDS, Alzheimer's disease, cancer and wound healingdisorders can be treated where inhibition or induction of apoptosis isindicated.

These, and further objects not explicitly referred to which however canwithout any difficulties be taken from the preceding appreciation of theprior art will be solved by the exemplified embodiments of the presentinvention defined in the claims.

This object is particularly solved by making available substancescomprising peptides having one of the amino acid sequences representedin SEQ ID NOs 1 through 19.

In a preferred exemplified embodiment of the present invention,apoptosis-inhibiting substances are concerned comprising amino acidsequences of the general formula (1):R-A₁—Y—V—V—M,wherein A₁ stands for A, D, E, G, M, N. T, W or Y, or pharmaceuticallyacceptable salts of these substances.

In a further preferred exemplified embodiment of the present invention,apoptosis-inducing substances are concerned comprising amino acidsequences of the general formula (2)R-A₂—Y—V—V-A₃,wherein A₂ stands for A, L, P, S or C and A₃, if A₂ stands for A, is Aand if A₂ stands for L, P, S or C, is M, or pharmaceutically acceptablesalts of these substances.

Further particularly preferred embodiments of the present inventioncomprise amino acid sequences shown in SEQ ID NOs 12, 18 and 19.

For the purposes of the present invention, the internationally-commonone-letter code for amino acids is used; i.e. A stands for alanine(Ala), C for cysteine (Cys), D for asparagine acid (Asp), E forglutaminic acid (Glu), F for phenyl alanine (Phe), G for glycine (Gly),L for leucine (Leu), M for methionine (Met), N for asparagine (Asn), Pfor prolin (Pro), R for arginine (Arg), S for serine (Ser), T forthreonine (Thr), V for valine (Val), W for tryptophane (Trp) and Y fortyrosine (Tyr). In this connection, L-amino acids are represented bycapital letters and D-amino acids by using small letters.

In accordance with a particularly preferred aspect, the presentinvention relates, therefore, to apoptosis-inhibiting substances,preferably proteins or peptides comprising one of the peptide sequencesshown sub SEQ ID NO 1 through SEQ ID NO 11, or the correspondingpharmaceutically acceptable salts thereof.

In accordance with a further particularly preferred aspect, the presentinvention relates also to apoptosis-inducing substances, preferablyproteins or peptides comprising one of the peptide sequences shown subSEQ ID NO 12 through SEQ ID NO 19, or the corresponding pharmaceuticallyacceptable salts thereof.

Furthermore, the present invention relates to the use of the substancesof the invention, preferably proteins or peptides comprising at leastone of the amino acid sequence shown in SEQ ID NO 1 through SEQ ID NO11, for the production of pharmaceuticals, particularly for theproduction of pharmaceuticals for the treatment of arteriosclerosis,AIDS and Alzheimer's disease and, particularly preferred in thisconnection, of arteriosclerosis.

The present invention relates, furthermore, to the use of the peptidesof the invention comprising at least one of the amino acid sequenceshown in SEQ ID NO 12 through SEQ ID NO 19, for the production ofpharmaceuticals, particularly for the production of pharmaceuticalswherein the death of cells should be caused, for instance for thetreatment of cancer.

Surprisingly, it was shown by the inventors that peptides comprising anamino acid sequence shown by formula (1) inhibit apoptosis at anextremely strong measure. In particular, these peptides areoutstandingly suited to inhibit flow-dependent TSP-1-induced apoptosisof endothelial cells. It is assumed, however, that these peptides arealso able to inhibit apoptosis in other cells.

As particularly preferred exemplified embodiments of the presentinvention, the following peptides/peptide sequences are madeavailable: 1. R-A-Y-V-V-M (SEQ ID NO 1) 2. R-W-Y-V-V-M (SEQ ID NO 2) 3.R-Y-Y-V-V-M (SEQ ID NO 3) 4. R-E-Y-V-V-M (SEQ ID NO 4) 5.K-R-A-Y-V-V-M-W-K-K (SEQ ID NO 5) 6. K-R-E-Y-V-V-M-W-K-K (SEQ ID NO 6)7. R-G-Y-V-V-M (SEQ ID NO 7) 8. R-M-Y-V-V-M (SEQ ID NO 8) 9. R-T-Y-V-V-M(SEQ ID NO 9) 10. R-N-Y-V-V-M (SEQ ID NO 10) 11. R-D-Y-V-V-M (SEQ ID NO11) 12. M-V-V-Y-F-R (SEQ ID NO 12) 13. R-A-Y-V-V-A (SEQ ID NO 13) 14.R-L-Y-V-V-M (SEQ ID NO 14) 15. R-P-Y-V-V-M (SEQ ID NO 15) 16.R-S-Y-V-V-M (SEQ ID NO 16) 17. R-C-Y-V-V-M (SEQ ID NO 17) 18.m-v-v-y-f-r (SEQ ID NO 18) 19. m-v-v-y-a-r (SEQ ID NO 19)

For the purposes of the present invention, there is understood under theterm “peptide” a substance consisting of a chain of 2 or more aminoacids bound by peptide bonds. In particular, apoptotically activepeptides according to the invention have a chain length of <100 aminoacids, preferably of <75, particularly preferred of <50, moreparticularly preferred of <25 and most preferred of <15 amino acids.

Particularly preferred for the purposes according to the invention are,therefore, peptides which comprise one of SEQ ID NOs 1 through 19 andcomprise either at the N-terminal and/or the C-terminal end 1 to 3additional amino acids each.

For the purposes of the present invention, the term “protein” will marka substance in which a plurality of “peptides” are bonded with oneanother, for instance by molecular bonds, such as disulfide bridges orby salt bridges. This definition covers simultaneously both nativeproteins and at least partly “artificial” proteins, while such“artificial” proteins can be altered, for instance by added chemicalresidues to the amino acid chain which do not occur in native proteins.

For the purposes of the present invention, the term “apoptoticallyactive” will mean that the addition of the respective substance to thetest system shown in examples 5 and 6 generates either a positive ornegative inhibition index. Particularly endothelial cells, preferablyespecially HUVEC, are to this end cultivated in a medium which includes1 μg TSP-1 per ml. The percentage apoptosis values of such positivecontrol will then serve, as shown in the examples, for the calculationof the inhibition index for those substances which are used as potentialinhibitors together with the inductor TSP-1 in parallel tests. Anegative control shows the absence of any other inductors.

A positive inhibition index shows in this connection that the respectivesubstance inhibits apoptosis. A negative inhibition index shows in thisconnection that the used substance induces apoptosis, i.e. amplifies theTSP-1-induced apoptosis.

Negative peptides, though, often show lower metabolic stabilityvis-a-vis peptidases and relatively small biological availability.

Starting from the above-shown peptides, one skilled in the art canwithout employing inventive activity develop quite a series of deductedcompounds which have a similar or equal mode of action and which arealso referred to, inter alia, as peptide mimetics.

For the purposes of the present invention, peptide mimetics will, inthis connection, stand for compounds which imitate the structure ofpeptides and, as ligands, are able to either imitate (agonist) or block(antagonist) the biological activity on the receptor/enzyme level. Thepeptide mimetics should in particular provide for improved biologicalavailability and improved metabolic stability. The kind of mimetizationmay extend from the lightly changed starting structure up to the purenon-peptide. See, for instance, A. Adang et al., Recl. Trav. Chim.Pays-Bas 113 (1994), 63-78.

In principle, the following possibilities formimetization/derivatization of a peptide structure are available:

-   -   Use of D- instead of L-amino acids    -   Modification of the side chain of amino acids    -   Alteration/lengthening of the peptide main chain    -   Cyclization for conformation stabilization    -   Use of templates enforcing a particular secondary structure    -   Use of a non-peptidic backbone which imitates together with        suitable residues/side chains the structure of the peptide.

While the proteolytic stability of a peptide can be increased byexchanging L- against D-amino acids, the modification of the side chainsof one of the amino acids often leads to an improvement of the bondingproperties of the whole peptide.

When changing the peptide backbone, an exchange of an amid group againstamid-like groupings occurs as a rule (J. Gante, Angew. Chem. 106 (1994),1780-1802). By these measures, both bonding affinity and metabolicstability of the native peptide can be influenced.

By cyclization of a linear peptide the flexibility thereof and hence itsglobal conformation is fixed. When fixing the biologically activeconformation, the affinity of the peptide relative to the receptor isincreased since entropy decrease when bonding is smaller than whenbonding a flexible linear peptide. To this end, amino acid side chainswhich do not take part in receptor recognition are connected with oneanother or with the peptide skeleton.

The secondary structure of the peptide plays a decisive role for themolecular recognition of the receptor. In addition to α-helix andβ-pleated sheet, so-called turns constitute significant conformationelements as turning points in the peptide chain. The replacement ofthese structural units by an element which stabilizes after theinsertion into a peptide a defined secondary structure, has led to theconcept of the secondary structure mimetic.

The water solubility, too, of the peptides can be increased for instanceby the introduction of S- and C-glycopeptide derivatives. Furthermeasures may for instance include polyethylene glycolization of thepeptides.

Lipophilicity of the hexapeptides may be increased as well in that forinstance phenyl alanines are attached to the peptide sequence.

Cyclization and N-terminal modification of peptides has for instancebeen described by Borchard, Journal of controlled Release 62 (1999),231-238, and by Blackwell et al., J. Org. Chem. 10 (2001), 5291-302.

It is, therefore, obvious that one skilled in the art starting from theknowledge imparted by the present invention will easily get to quite aseries of deducted peptide mimetics which, however, are all covered bythe scope of the present invention.

Under a further preferred aspect, the present invention provides alsofor peptide mimetics which have been deducted from SEQ ID NOs 1 through19 and for substances comprising such peptide mimetics.

In particular, the present invention provides, under a further preferredaspect, the use of peptide mimetics deducted from SEQ ID NOs 1 through11 and of substances comprising such peptide mimetics for the productionof a pharmaceutical for treating arteriosclerosis, AIDS and Alzheimer'sdisease and, in this connection, with preference of arteriosclerosis.

In particular, the present invention provides, under a further preferredaspect, the use of peptide mimetics deducted from SEQ ID NOs 12 through19 and substances comprising such peptide mimetics for the production ofa pharmaceutical for cancer treatment.

Under a further preferred aspect of the present invention, suchpeptides, or correspondingly derivatized peptides, respectively, canalso be used as diagnostic reagents. As shown be the inventors, thesubstances of the invention, preferably the peptides of SEQ ID NOs 1through 19, bind to apoptotic cells and can, therefore, outstandingly beused as a diagnosis tool, for instance to prove the existence ofapoptotic cells in the area of arteriosclerotic lesions and hencelesions as such. To this end, the substances are labeled. For suchlabeling, one skilled in the art is aware of a plurality of methodswhich can be selected according to the purpose of use.

Suitable methods have for instance been described in US patentSpecifications U.S. Pat. No. 4,479,930 (Hnatovich), U.S. Pat. No.4,652,440 (Paik et al.) and U.S. Pat. No. 4,668,503 (Hnatowich).

For the purposes of the present invention, the term “labeled substanceaccording to the invention” denotes any substance of the invention whichincludes labeling substances known from the prior art and used as astandard for labeling peptides, such as general radio isotopes such asfor instance rhenium or technetium, but also enzymes, enzyme substrates,antibodies, epitopes for the recognition on specificantibodies/fragments etc. One skilled in the art will very easilyrecognize to above enumeration as exemplary and not conclusive.

If the substances according to the invention are to be used as diagnosistool, any labeling method known in the prior art can be used.

Substances according to the invention which constitute active componentsof a pharmaceutical preparation are, in general, dissolved in apharmaceutically acceptable carrier. Examples of pharmaceuticallyacceptable carriers may include buffer solutions such as phosphatebuffer or citrate buffer. In order to maintain the activity of thepeptides, reagents may also be added which are pharmaceuticallyacceptable maintaining, for instance, a reducing environment in thepharmaceutical preparation.

The specific dosage and posology depends for each patient on a number offactors including the activity of the used specific compounds, thepatient's age, the body weight, the general health status, the gender,nutrition, time of the administration, way of administration, theexcretion rate, the combination with other pharmaceuticals and theseverity of the individual disease to which the therapy is applied.Depending on these factors, it is determined by a physician.

Normally, peptide pharmaceuticals are parenterally administered, forinstance by an inhalation spray, rectally, by subcutaneous, intravenous,intramuscular, intra-articular and intrathecal injection and infusiontechniques, or externally in pharmaceutical formulations which includeconventional pharmaceutically acceptable carriers, adjuvants andvehicles. Depending on the kind of the identified substance, other waysof administration, oral for instance, can be considered as well. Inwound healing, the identificates according to the invention arepreferably administered in the form of ointments or powder.

The present invention also provides for pharmaceutical compositionswhich comprise an active amount of an apoptotically active substance,preferably a peptide, a protein or peptide mimetic in combination with aconventional pharmaceutical carrier. A pharmaceutical carrier is forinstance a solid or liquid filler, an encapsulation material or asolvent. Examples of materials which can serve as pharmaceuticalcarriers include sugars, such as lactose, glucose and saccharose; starchsuch as maize starch and potato starch, cellulose and the derivativesthereof, such as sodium carboxymethyl cellulose, ethylcellulose andcellulose acetate; pulverized tragacanth; malt, gelatin, tallow;pharmaceutical carriers such as cocoa butter and suppository wax; oilssuch as peanut oil, cotton seed oil, carthamus oil, sesame oil, oliveoil, maize oil, soy bean oil; polyalcohols, such as propylene glycol,glycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laureate; agar, buffer means, such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogenically-freewater; isotonic salt solution; Ringer's solution, ethyl alcohol andphosphate buffer solutions, as well as other non-toxic compatiblesubstances which are used in pharmaceutical formulations. Wettingagents, emulgators and lubricating agents, such as sodium lauryl sulfateand magnesium stearate, also coloring agents, coating agents andperfuming agents and preserving agents can be present in thepreparations as well, corresponding to the galenic specialist's request.The amount of the active agent which is combined with the carriermaterials in order to produce an individual dose will vary depending onthe patient to be treated and the particular method of administration.

Pharmaceutically acceptable salts of the substances according to theinvention, preferably peptides, proteins or peptide mimetics can beproduced in a well known way, for instance by dissolving the compoundsof the invention in the corresponding diluted acid or base, e.g.hydrochloric acid or sodium hydroxide solution, and subsequentlyfreeze-drying them. Metal salts can be obtained by dissolving thecompounds according to the invention in solutions which contain thecorresponding ion, and subsequently isolating the compound via HPLC orgel permeation processes.

The following examples will explain the invention in more detail:

EXAMPLE 1 Cultivation of Human Umbilical Vein Endothelial Cells (HUVEC)

Solutions (sterile):

-   -   Culture medium: IF basal medium+15% (v/v) NCS; 5 μg/ml        transferrin, 5 μg/ml heparin, 0.7 μg/ml FGF, 2 mM L-glutamine        [IF basal medium: 1:1 mixture of Iscove's Modified Dulbecco's        Medium (IMDM) and Ham's F12, both produced by Life Technologies,        Paisley (England)]    -   NCS: Newly-born calves serum (Sebak, Aidenbach)    -   FGF: Fibroblast growth factor (own production, purified from pig        brain).        Materials:

Cell culture vessels, gelatinized

Procedure:

Cultivation of HUVEC takes place in gelatin-coated culture vessels at37° C., 5% CO₂ and water vapor saturated atmosphere. The culture mediumis exchanged every 2 to 3 days; in case of confluence, the cells arepassaged at a cleavage rate of from 1:3 to 1:5. HUVEC grow strictlycontact-inhibited and form unilaminar cell bed having the typicalcobble-stone morphology. In case of confluence, the cultures reach celldensities of 4-9×1 cells/cm². For apoptosis examinations, HUVEC culturesof passages 1-4 are exclusively employed.

Coating of Culture Vessels:

Solutions (sterile):

-   -   Gelatin solution, 1% (w/v) in milli-Q-water    -   Suspend 1 g gelatin (cell culture tested) in 100 ml        milli-Q-water, dissolve by autoclaving for 20 minutes at 121° C.        and 2 bars, and store at room temperature.    -   PBS (140 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄)    -   8 g/l NaCl    -   0.2 g/l KCl    -   1.44 g/l Na₂HPO₄×2H₂O    -   0.2 g/l KH₂PO₄    -   Dissolve the salts in a corresponding volume of milli-Q-water,        autoclave for 20 minutes at 121° C. and 2 bars, and store at        room temperature. The pH value is controlled and is between 7.2        and 7.4.        Materials:

Cell culture vessels

Procedure:

For the cultivation of adherently growing cells, culture vessels arecoated with gelatin. The bottom of the cell culture vessels is coveredwith sterile gelatin solution, the cell culture vessels are maintainedfor 15 minutes at room temperature. The gelatin solution is sucked off,the cell culture vessels are once washed with PBS and in such a statecan be used.

Subcultivation of Adherent Cells

Solutions (sterile):

-   -   PBS    -   Trypsin/EDTA solution    -   Dissolve 0.05% (w/v) trypsin and 0.02 (w/v) EDTA in PBS and        filter aseptically.        Materials:

Cell culture vessels, gelatinized

Procedure:

The cells are detached with a Trypsin/EDTA solution from the culturevessel. The culture medium is sucked off, the bottom of the culturevessel is shortly washed with PBS and covered with Trypsin/EDTA solution(˜1 ml for a culture face of 25 cm²). The enzyme solution is immediatelysucked off again so that a thin liquid film remains on the cells. Thecells are maintained for 1 to 10 minutes at room temperature and thedetachment of the cells is observed under the microscope. Detachment ofthe cells can be accelerated by gently pounding the culture vessel atthe edge. The cells are received in a fresh culture medium, possiblycounted, and seeded in new culture vessels.

EXAMPLE 2 Determination of the Apoptosis Rate by Dyeing Apoptotic Cellswith DAPI

DAPI belongs to the indole dye stuff group and is a selective DNS dyestuff. The dye stuff is activated et 340-360 nm, and the emissionmaximum is at about 480 nm. It is used for apoptosis examinations[compare Cohen et al. Immunology Today, 14. No. 3, 126-130 (1993)]

Morphologic Evaluation:

Solutions:

-   -   PBS    -   Formaldehyde solution    -   4% (v/v) formaldehyde in PBS    -   DAPI solution (Molecular Probes, Leiden, Netherlands)    -   2 μg/ml DAPI in methanol        Materials:

Petri dish (35 mm) with cells in culture

Procedure:

The culture supernatant of a Petri dish is sucked off, the cell bed isfixed on ice for 15 minutes with 1 ml formaldehyde solution, is washedtwice with 2 ml PBS, is added 0.5 ml DAPI solution for 15 minutes, iswashed with PBS and evaluated under the fluorescence microscope. A UVfilter set and a 20x and a 40x objective are used. 500 to 1000 cells areselected at random and the cells having apoptotic nuclei are counted.

The apoptosis index is calculated in accordance with the followingformula:Apoptosis index[%]=number of apoptotic cells/total cell number×100Flow Cytometry:Solutions:

-   -   PBS    -   Medium    -   Ethanol (reagent-grade) ice-cooled (−20° C.)    -   DAPI buffer    -   DAPI stock solution    -   DAPI dye solution        Procedure:

The culture supernatant is sucked off and the cells are trypsinizedwithout washing them with PBS. The cell suspension is received inmedium, is counted, is centrifuged at 800×g for 5 minutes, the sedimentis re-suspended in 0.5 ml IF and is dripped into 1.5 ml ice-coldethanol. The suspension is stored overnight at 20° C. After renewedcentrifugation and re-suspending of the sediment in 2 ml PBS, therefollows a half-hour incubation at 37° C., further centrifugation,re-suspending of the sediment in 5 ml DAPI solution, and counting in theflow cytometer at a counting rate of 50 to 300 events per second. Thecoating obtained shows a high peak of cells in the G phase of the cellcycle, followed by a fraction of cells in the S phase (mediumfluorescence intensities) and a last peak of high fluorescenceintensities representing the cells in the G₂ phase. Caused by thedecreasing absolute DNS amount per cell, apoptotic cells appear in asub-G₁ peak [Darzynklewicz Z. et al., Cytometry, 13, 795-808 (1992);Zamai L. et al., Cytometry, 14, 891-897 (1993]. This shows theappearance of an apoptosis under the selected conditions.

EXAMPLE 3 Induction of Apoptosis and Test System for ApoptoticallyActive Peptides or Proteins in Case of Cultivated Endothelial Cells

The cells are cultivated as described in Example 1. Having obtainedcomplete confluence, the cells are used for the test. First,thrombospondin 1 (1 μg/ml) is added to fresh medium and compared to theeffect of fresh medium on the apoptosis rate of HUVEC. Table 1 showsthat the addition of thrombospondin leads to a significant increase ofthe apoptosis rate. The apoptosis observed while adding fresh medium isinduced by the thrombospondin secreted during the course of theexperiment. TABLE 1 Induction of apoptosis at HUVEC with TSP-1 Culturemedium Apoptosis rate (%) after 24 h Fresh medium 0.9 ± 0.1 Fresh +TSP-1 (1 μg/ml) 3.0 ± 0.4

EXAMPLE 4 Peptide Synthesis

The peptides used were synthetisized by the NMI (Naturwissenschaftlichesund Medizinisches Institut an der Universitat Tuebingen, Reutlingen,Germany) based on data provided by Cyto Tools.

EXAMPLE 5 Identification of Apoptotically Active Hexamer Peptides Basedon the Process According to the Present Invention

The cells were cultivated as described in Example 1. The cells areseeded in the corresponding culture vessels (e.g. 24-hole plate/0.5 mlper cavity) and, having obtained complete confluence, are employed forthe test. The cells are provided with new medium:

-   (a) fresh culture medium [Base rate of apoptosis],-   (b) fresh medium with 1 μg/ml TSP-1 [Apoptosis-induced substance;    control],-   (c) medium (b)+peptide of SEQ ID NO 1, 1 mM-   (d) medium (b)+peptide of SEQ ID NO 2, 1 mM-   (e) medium (b)+peptide of SEQ ID NO 3, 1 mM-   (f) medium (b)+peptide of SEQ ID NO 4, 1 mM-   (g) medium (b)+peptide of SEQ ID NO 7, 1 mM-   (h) medium (b)+peptide of SEQ ID NO 8, 1 mM-   (i) medium (b)+peptide of SEQ ID NO 9, 1 mM-   (j) medium (b)+peptide of SEQ ID NO 10, 1 mM-   (k) medium (b)+peptide of SEQ ID NO 11, 1 mM-   (l) medium (b)+peptide of SEQ ID NO 12, 1 mM-   (m) medium (b)+peptide of SEQ ID NO 13, 1 mM-   (n) medium (b)+peptide of SEQ ID NO 14, 1 mM-   (o) medium (b)+peptide of SEQ ID NO 15, 1 mM-   (p) medium (b)+peptide of SEQ ID NO 16, 1 mM-   (q) medium (b)+peptide of SEQ ID NO 17, 1 mM-   (r) medium (b)+peptide of SEQ ID NO 18, 1 mM-   (s) medium (b)+peptide of SEQ ID NO 19, 1 mM

After 24 hours of incubation under culture conditions (Example 1), thecells are fixed, dyed with DAPI and morphologically examined under thefluorescence microscope, or flow-cytometricaily examined, respectively.The apoptotic cells and the total number are determined and theapoptosis index is calculated (percentage of apoptotic cells). The dataof 3 independent experiments giving the mean values and the standarddeviation are given in Table 2. Inhibitorically active peptides show, inaccordance with the invention, a positive inhibition index whileinductory peptides show, in accordance with the invention, a negativeinhibition index. TABLE 2 The following peptides are tested: AminoApoptosis Inhibition SEQ ID NO acid sequence index [%] index [%]* KControl 3.28 ± 0.55 (1) R-A-Y-V-V-M 0.83 ± 0.24 +75.3 ± 7.5 (2)R-W-Y-V-V-M 1.30 ± 0.26 +61.9 ± 7.7 (3) R-Y-Y-V-V-M 0.85 ± 0.17 +74.7 ±5.0 (4) R-E-Y-V-V-M 1.04 ± 0.30 +69.0 ± 8.9 (7) R-G-Y-V-V-M 1.83 ± 0.37 +46.4 ± 11.1 (8) R-M-Y-V-V-M 1.84 ± 0.39  +44.2 ± 11.6 (9) R-T-Y-V-V-M1.86 ± 0.36 +44.5 ± 9.8 (10) R-N-Y-V-V-M 2.06 ± 0.53  +39.5 ± 15.8 (11)R-D-Y-V-V-M 2.12 ± 0.47  +37.3 ± 14.0 (12) M-V-V-Y-F-R 4.40 ± 0.53 −34.1± 6.2 (13) R-A-Y-V-V-A 4.98 ± 0.3   −51.8 ± 6.0 (14) R-L-Y-V-V-M 6.41 ±0.63 −95.4 ± 6.1 (15) R-P-Y-V-V-M 5.67 ± 0.54 −72.8 ± 6.4 (16)R-S-Y-V-V-M 6.86 ± 0.37 −108.4 ± 3.7  (17) R-C-Y-V-V-M 13.97 ± 0.35 −326.5 ± 3.6  (18) m-v-v-y-f-r 14.6 ± 0.75 −345.1 ± 5.1  (19)m-v-v-y-a-r 11.4 ± 0.63 −247.1 ± 5.4 *positive inhibition index = substance inhibited; negative inhibitionindex = substance induced;

According to the invention, the inhibition index is calculated asfollows:

-   -   inhibition index [%]=(measured apoptosis index·100/apoptosis        index control)−100

EXAMPLE 6 Improvement of the Inhibition Effect by Lengthening the AminoAcid Chain

Cell cultivation and the tests were carried out as described in Example5. The peptides used are enumerated in Table 3. TABLE 3 Results withlengthened peptide sequences Apoptosis Inhibition SEQ ID NO Amino acidsequence index [%] index [%]* Control 8.73 ± 0.43 (5)K-R-A-Y-V-V-M-W-K-K 0.37 ± 0.22 +95.8 ± 2.6 (6) K-R-E-Y-V-V-M-W-K-K 0.48± 0.22 +94.5 ± 2.5*positive inhibition index = substance inhibited; negative inhibitionindex = substance induced

1. Apoptotically active substance comprising at least one peptideportion having one of the amino acid sequences shown in SEQ ID NOs 1through
 19. 2. Substance according to claim 1, comprising a peptide orprotein.
 3. Substance according to claim 1 selected from the groupconsisting of the peptides shown in SEQ ID NO 1 through SEQ ID NO
 19. 4.Substance according to claim 1, comprising a peptide mimetic deductedfrom one of the amino acid sequences shown in SEQ ID NO 1 through SEQ IDNO
 19. 5. Apoptotically active substance according to claim 1,comprising at least one peptide portion having one of the amino acidsequences shown in SEQ ID NOs 1 through 11 or a peptide mimetic deductedtherefrom and is anti-apoptotically active.
 6. Apoptotically activesubstance according to claim 1, comprising at least one peptide portionhaving one of the amino acid sequences shown in SEQ ID NOs 12 through 19or a peptide mimetic deducted therefrom and is apoptosis-inducinglyactive.
 7. (canceled)
 8. A pharmaceutical preparation comprising asubstance according to claim 1 or a pharmaceutically acceptable saltthereto dissolved in a pharmaceutically acceptable.
 9. Method for thetreatment of arteriosclerosis comprising administering a pharmaceuticalcomposition according to claim 18 to a patient in need of suchtreatment.
 10. Method for the treatment of Alzheimer's diseasecomprising administering a pharmaceutical composition according to claim18 to a patient in need of such treatment.
 11. Method for the treatmentof AIDS comprising administering a pharmaceutical composition accordingto claim 18 to a patient in need of such treatment.
 12. Method for thetreatment of wounds comprising administering a pharmaceuticalcomposition according to claim 18 to a patient in need of suchtreatment.
 13. Method for the treatment of cancer comprisingadministering a pharmaceutical preparation according to claim 19 to apatient in need of such treatment.
 14. Substance according to claim 1,which is labeled.
 15. Method of diagnosis comprising binding a substanceaccording to claim 14 to apoptotic cells.
 16. Method according to claim15 for the detection of arteriosclerotic lesions comprising binding saidsubstance to apoptotic cells in the area of said lesions.
 17. Methodaccording to claim 15 for cancer comprising binding said substance toapoptotic cells in the area of said cancer.
 18. A pharmaceuticalcomposition comprising a substance according to claim 5 or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier.
 19. A pharmaceutical composition comprising asubstance according to claim 6 or a pharmaceutically acceptable saltthereof and a pharmaceutically acceptable carrier.